Novel molecular imaging approaches to monitor gene and cell-based therapies Gene and cell-based therapies have ushered in a new era of opportunities in regenerative medicine and oncology. However, a critical roadblock in the effective development and evaluation of cellular therapeutics is the inability to follow the fate and function of the therapeutic genes and cells in treated patients. We propose to develop technologies for specific identification and tracking of therapeutic genes and cells in vivo using positron emission tomography (PET), a quantitative, non-invasive molecular imaging approach applicable to both preclinical and clinical settings. This application addresses a key limitation of current reporter gene strategies, in which therapeutic vectors and cells are genetically modified to produce a signal detectable by PET. Instead of commonly used, highly immunogenic viral proteins, we will generate novel PET reporter genes based on fully human proteins, to overcome this challenge to clinical implementation. This project relies on many years of grants and basic research results that are now ready to advance to the commercial domain. We propose a three year effort to turn recent advances into practical outcomes delivered as end-user-ready PET Reporter Gene (PRG) delivery kits and PET Reporter Probes (PRP) that will enable whole body pharmacokinetic and therapeutic outcomes information. This application will also deliver preliminary information from a first-in-human Phase 0 small trial of new PET reporter probe biodistribution and dosimetry. Our proposal leverages an established partnership between UCLA (the laboratories of the Ahmanson Translational Imaging Division and the laboratory of Harvey Herschman) and CellSight Technologies (CST, a biotech company based in San Francisco, CA). The UCLA-CST partnership builds on past extensive interactions at UCLA between investigators and consultants, as described in this application. We will carry out four Specific Aims. Aim 1 consists of in vitro, cell culture and animal studies to evaluate and optimize new PET Reporter Gene-PET Reporter Probe (PRG-PRP) systems. Our new current PRG being developed is a point mutant (N44D) of the human thymidine kinase 2 (tk2) gene. L-[18F]FMAU and L- [18F]FEAU, two hTK2-N44D substrates, are our new PRPs. In Aim 1 we will also determine whether the TK2- based PRG can elicit an immune response in humans and we propose a strategy to eliminate this possibility. In Aim 2 we propose a stringent preclinical evaluation of the new PRG-PRP systems, using animal models of gene and cell-based therapies against two types of cancer: hepatic metastases of colorectal cancer and melanoma. Aim 3 proposes a strategy to develop, validate, and commercialize kits for PRG delivery into murine and human therapeutic cells and a plan to disseminate this new capability to wider communities of end- users. In Aim 4 we will complete an eIND submission to enable first-in-human studies of the biodistribution and dosimetry of the new PET reporter probes L-FMAU and L-FEAU. These "first-in-human" studies will set the stage for a follow-up study in which UCLA and CST will submit a full IND application to the FDA to initiate clinical testing of the new PRG-PRP systems in cancer patients. The set of new PET imaging technologies co-developed by UCLA and CST investigators may find immediate clinical applications in experimental gene and cell-based therapies in cancer and may be broadly applicable to therapies for diseases with significant public health impact, including transplantation of hematopoietic stem cells in congenital and acquired disorders such as AIDS, islet cells in type 1 diabetes, ES-derived neural stem cells in Parkinson's disease, and stem cells in myocardial dysfunction. PUBLIC HEALTH RELEVANCE: One of the great promises of cell-based therapies is that physicians will find a way to isolate and modify patient's stem cells or T lymphocytes so that they can be re-injected into patients to treat their disease. However, a key challenge is to be able to monitor the cells after they have been administered and see if they survive and engraft, whether they home to areas of disease, and whether they are able to reestablish the activity needed to counteract disease. We are developing novel tools to follow the fate and function of transplanted cells, based on a powerful medical camera called the PET scanner. PET imaging, or positron emission tomography, allows doctors to visualize the biology of cells in living organisms, including patients. The development of novel tools and technologies that will ultimately enable the routine use of PET for clinical monitoring of cell-based therapies in oncology and regenerative medicine represents a complex endeavor that exceeds the capabilities of a typical academic group and carries substantial risks for start-up biotech companies. In our opinion, the solution is an integrated approach in which academic and industry partners work together from the inception of the project, on both discovery and product development phases. The scientific yield from this work may lead to improved therapies for cancer, significantly impacting public health.